The key virulence determinants and protective immunogens of the syphilis agent, Treponema pallidum, have yet to be elucidated, largely due to the inability of researchers to continuously cultivate this organism in vitro. Recent studies have indicated that the T. pallidum outer membrane is a relatively protein-deficient, antigenically inert structure that may be a major factor in the ability of this pathogen to evade host immune defenses. While this membrane appears to be devoid of major protein species, freeze fracture studies have shown that it does contain an extremely small number of integral protein molecules, at least some of which are surface exposed, and recent studies strongly suggest that these same outer membrane proteins are a key target of treponemicidal antibodies. In addition, our laboratories have demonstrated that T. pallidum synthesizes, also in very small quantities, a number of low molecular weight proteins that are localized beyond the outer membrane. Such extracellular proteins also must be considered important virulence factors and/or immunogens of T. pallidum. In order to assess the roles of these cell surface and extracellular proteins in the pathogenesis and immunobiology of syphilis, it seems clear that their structural genes must be cloned and expressed in a cultivable organism such as Escherichia coli. For the project period, we propose to do the following: (i) A T pallidum genomic library will be constructed using a high copy E. coli vector which will promote the expression of treponemal proteins at an elevated level. (ii) Clones encoding treponemal proteins synthesized with amino-terminal signal peptides characteristic of cell surface and extracellular proteins will be identified by using TnphoA, a transposon specifically designed to search for such export signals. (iii) Candidate recombinant proteins will be screened for additional properties expected for T. pallidum surface and extracellular proteins, their native treponemal counterparts will be identified and localized, and their vaccinogenic potential will be assessed. In view of the paucity of information regarding the physiology of this noncultivable spirochete, these studies should provide useful information and nicely complement our other ongoing studies.